Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• (3)Birch, R. R., Biver, C., Campagna, R., Gledhill, W.E., Pagga,U., Steber, J., Reust, H. and Bontinck, W.J. (1989) Screening of chemicals for anaerobic biodegradation. Chemosphere 19, 1527-1550. (Also published as ECETOC Technical Report No. 28, June 1988).

• (4)Shelton D.R. and Tiedje, J.M. (1984) General method for determining anaerobic biodegradation potential. Appl. Environ. Mircobiology, 47, 850-857.

• (5)Owen, W.F., Stuckey, DC., Healy J.B., Jr, Young L.Y. and McCarty, P.L. (1979) Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res. 13, 485-492.

• (6)Healy, J.B.Jr. and Young, L.Y. (1979) Anaerobic biodegradation of eleven aromatic compounds to methane. Appl. Environ. Microbiol. 38, 84-89.

• (7)Gledhill, W.E. (1979) Proposed standard practice for the determination of the anaerobic biodegradation of organic chemicals. Working document. Draft 2 no.35.24. American Society for Testing Materials, Philadelphia.

• (8)Battersby, N.S. and Wilson, V. (1988) Evaluation of a serum bottle technique for assessing the anaerobic biodegradability of organic chemicals under methanogenic conditions. Chemosphere, 17, 2441-2460.

• (9)E1192-92. Standard Test Method for Determining the Anaerobic Biodegradation Potential of Organic Chemicals. ASTM, Philadelphia.

• (10)US-EPA (1998) Fate, Transport and Transformation Test Guidelines OPPTS 835.3400 Anaerobic Biodegradability of Organic Chemicals.

• (11)International Organization for Standardization (1995) ISO 11 734 Water Quality — Evaluation of the ultimate anaerobic biodegradation of organic compounds in digested sludge — Method by measurement of the biogas production.

• (12)International Organization for Standardization (2003) ISO 13 641-1 Water Quality — Determination of inhibition of gas production of anaerobic bacteria — Part 1 General Test.

• (13)International Organization for Standardization (1995) ISO 10 634 Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium.

• (14)Pagga, U. and Beimborn, D.B., (1993) Anaerobic biodegradation test for organic compounds. Chemosphere, 27, 1499-1509.

- (15)International Organization for Standardization (1997) ISO 11 923 Water Quality — Determination of suspended solids by filtration through glass-fibre filters.

Appendix 1

Example of an apparatus to measure biogas production by gas pressure

Image omitted; see official source

Key:

1	—	Pressure meter
---	---	---
2	—	3-way gas-tight valve
---	---	---
3	—	Syringe needle
---	---	---
4	—	Gastight seal (crimp cap and septum)
---	---	---
5	—	Head space (Vh)
---	---	---
6	—	Digested sludge inoculum (Vl)

Test vessels in an environment of 35 °C ± 2 °C

Appendix 2

Conversion of the pressure-meter

The pressure-meter readings may be related to gas volumes by means of a standard curve produced by injecting known volumes of air at 35 °C ± 2 °C into serum bottles containing a volume of water equal to that of the reaction mixture, V R:

• — Dispense V R ml aliquots of water, kept at 35 °C ± 2 °C into five serum bottles. Seal the bottles and place in a water bath at 35 °C for 1 hour to equilibrate;

• — Switch on the pressure-meter, allow to stabilise, and adjust to zero;

• — Insert the syringe needle through the seal of one of the bottles, open the valve until the pressure meter reads zero and close the valve;

• — Repeat the procedure with the remaining bottles;

• — Inject 1 ml of air at 35 °C ± 2 °C into each bottle. Insert the needle (on the meter) through the seal of one of the bottles and allow the pressure reading to stabilise. Record the pressure, open the valve until the pressure reads zero and then close the valve;

• — Repeat the procedure for the remaining bottles;

• — Repeat the total procedure above using 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 8 ml, 10 ml, 12 ml, 16 ml, 20 ml and 50 ml of air;

- — Plot a conversion curve of pressure (Pa) against gas volume injected Vb (ml). The response of the instrument is linear over the range 0 Pa to 70 000 Pa, and 0 ml to 50 ml of gas production.

Appendix 3

Example of a degradation curve (cumulative net pressure increase)

Image omitted; see official source

Text of image

Gas pressure (m bar)

Time (days)

Plateau

Lag phase

60

50

40

30

20

10

0

0

100

200

300

400

500

Appendix 4

Example of data sheets for the anaerobic biodegradation test — Data sheet for the test substance

Laboratory: …	Laboratory: …	Laboratory: …	Laboratory: …	Laboratory: …	Test substance: …	Test substance: …	Test substance: …	Test substance: …	Test No.: …	Test No.: …	Test No.: …	Test No.: …
Test temperature: (°C): …	Test temperature: (°C): …	Test temperature: (°C): …	Test temperature: (°C): …	Test temperature: (°C): …	Volume of headspace (Vh): …(l)	Volume of headspace (Vh): …(l)	Volume of headspace (Vh): …(l)	Volume of headspace (Vh): …(l)	Volume of liquid (Vl ): …(l)	Volume of liquid (Vl ): …(l)	Volume of liquid (Vl ): …(l)	Volume of liquid (Vl ): …(l)
Carbon in test substance Cc,v: …(mg/l)	Carbon in test substance Cc,v: …(mg/l)	Carbon in test substance Cc,v: …(mg/l)	Carbon in test substance Cc,v: …(mg/l)	Carbon in test substance Cc,v: …(mg/l)	mv [^E0220]: …(mg)	mv [^E0220]: …(mg)	mv [^E0220]: …(mg)	mv [^E0220]: …(mg)
Day	p 1 (test) (mbar)	p 2 (test) (mbar)	p 3 (test) (mbar)	p (test) mean (mbar)	p 4 (blank) (mbar)	p 5 (blank) (mbar)	p 6 (blank) (mbar)	p (blank) mean (mbar)	p (net) test — blank mean (mbar)	Δp (net) Cumulative (mbar)	mh headspace C [^E0221] (mg)	Dh Biodegradation [^E0222] (%)
	C IC, 1 test (mg)	C IC, 2 test (mg)	C IC, 3 test (mg)	C IC test mean (mg)	C IC, 4 blank (mg)	C IC, 5 blank (mg)	C IC, 6 blank (mg)	C IC blank mean (mg)	C IC, net test -blank mean (mg)	m l liquid C [^E0223] (mg)	m t total C [^E0224] (mg)	D t Biodegradation [^E0225] (%)
IC (end)
pH (end)

Carbon in test vessel, m v (mg): m v = C C,v×Vl

Carbon in headspace, m h (mg) at normal incubation temperature (35 °C): m h = 0,468 Δp × Vh

Biodegradation calculated from headspace gas, D h (%): D h = (m h × 100)/m v

Carbon in liquid, m l (mg): m l = C IC,net × Vl

Total gasified carbon, mt (mg): mt + m l

Total biodegradation, Dt (%): Dt = (mt × 100)/m v

Laboratory: …	Laboratory: …	Laboratory: …	Laboratory: …	Laboratory: …	Reference substance: …	Reference substance: …	Reference substance: …	Reference substance: …	Test No.: …	Test No.: …	Test No.: …	Test No.: …
Test temperature: (°C): …	Test temperature: (°C): …	Test temperature: (°C): …	Test temperature: (°C): …	Test temperature: (°C): …	Volume of headspace (Vh): …(l)	Volume of headspace (Vh): …(l)	Volume of headspace (Vh): …(l)	Volume of headspace (Vh): …(l)	Volume of liquid (Vl) (litres): …	Volume of liquid (Vl) (litres): …	Volume of liquid (Vl) (litres): …	Volume of liquid (Vl) (litres): …
Carbon in reference substance C c,v (mg/l): …	Carbon in reference substance C c,v (mg/l): …	Carbon in reference substance C c,v (mg/l): …	Carbon in reference substance C c,v (mg/l): …	Carbon in reference substance C c,v (mg/l): …	mv [^E0226] (mg):	mv [^E0226] (mg):	mv [^E0226] (mg):	mv [^E0226] (mg):
Day	p 1 (ref.) (mbar)	p 2 (ref.) (mbar)	p 3 (ref.) (mbar)	p (ref.) mean (mbar)	p 4 (inhib.) (mbar)	p 5 (inhib.) (mbar)	p 6 (inhib.) (mbar)	p (inhib.) mean (mbar)	p (ref.) ref. — blank (mbar)	Δp (ref.) cumulative (mbar)	m h headspace C [^E0227] (mg)	D h Biodegradation [^E0228] (%)
	C IC, 1 ref. (mg)	C IC, 2 ref. (mg)	C IC, 3 ref. (mg)	C IC ref. mean (mg)	C IC, 4 inhib. (mg)	C IC, 5 inhib. (mg)	C IC, 6 inhib. (mg)	C IC inhib. mean (mg)	C IC, net ref. — inhib. (mg)	m l liquid C [^E0229] (mg)	m t total C [^E0230] (mg)	D t Biodegradation [^E0231] (%)
IC (end)
pH (end)

Carbon in test vessel, m v (mg): m v = C C,v × Vl

Carbon in headspace, m h (mg) at normal incubation temperature (35 °C): m h = 0,468 Δp × Vh

Biodegradation calculated from headspace gas, D h (%): D h = (m h × 100)/m v

Carbon in liquid, m l (mg): m l = C IC,net × Vl

Total gasified carbon, mt (mg): mt + m l

[^E0231]: (⁶)

Total biodegradation, Dt (%): Dt = (mt × 100)/m v

C.44.   LEACHING IN SOIL COLUMNS

INTRODUCTION

• 1.This test method is equivalent to OECD Test Guideline (TG) 312 (2004). Man-made chemicals may reach soil directly via deliberate application (e.g. agrochemicals) or via indirect routes (e.g. via waste water → sewage sludge → soil or air → wet/dry deposition). For risk assessment of these chemicals, it is important to estimate their potential for transformation in soil and for movement (leaching) into deeper soil layers and eventually into groundwater.

• 2.Several methods are available to measure the leaching potential of chemicals in soil under controlled laboratory conditions, i.e. soil thin-layer chromatography, soil thick-layer chromatography, soil column chromatography, and adsorption — desorption measurements (1)(2). For non-ionised chemicals, the n-octanol-water partition coefficient (Pow) allows an early estimation of their adsorption and leaching potential (3)(4)(5).

• 3.The method described in this test method is based on soil column chromatography in disturbed soil (see Appendix 1 for definition). Two types of experiments are performed to determine (i) the leaching potential of the test chemical, and (ii) the leaching potential of transformation products (study with aged residues) in soils under controlled laboratory conditions ([^E0232]). The test method is based on existing methods (6)(7)(8)(9)(10)(11).

- 4.An OECD Workshop on soil/sediment selection, held at Belgirate, Italy in 1995 (12) agreed on the number and type of soils for use in this test method. It also made recommendations with regard to collection, handling and storage of soil samples for leaching experiments.

PRINCIPLE OF THE TEST METHOD

- 5.Columns made of suitably inert material (e.g. glass, stainless steel, aluminium, teflon, PVC, etc.) are packed with soil and afterwards saturated and equilibrated with an ‘artificial rain’ solution (for definition see Appendix 1) and allowed to drain. Then the surface of each soil column is treated with the test chemical and/or with aged residues of the test chemical. Artificial rain is then applied to the soil columns and the leachate is collected. After the leaching process the soil is removed from the columns and is sectioned into an appropriate number of segments depending on the information required from the study. Each soil segment and the leachate are then analysed for the test chemical and, if appropriate, for transformation products or other chemicals of interest.

APPLICABILITY OF THE TEST METHOD

- 6.The test method is applicable to test chemicals (unlabelled or radio-labelled: e.g. ¹⁴C) for which an analytical method with sufficient accuracy and sensitivity is available. The test method should not be applied to chemicals which are volatile from soil and water and thus do not remain in soil and/or leachate under the experimental conditions of this test method.

INFORMATION ON THE TEST CHEMICAL

• 7.Unlabelled or radio-labelled test chemicals can be used to measure the leaching behaviour in soil columns. Radio-labelled material is required for studying the leaching of transformation products (aged residues of the test chemical) and for mass balance determinations. ¹⁴C-labelling is recommended but other isotopes, such as ¹³C, ¹⁵N, ³H, ³²P, may also be useful. As far as possible, the label should be positioned in the most stable part(s) of the molecule. The purity of the test chemical should be at least 95 %.

• 8.Most chemicals should be applied as single substance However, for active substances in plant protection products, formulated products may be used to study the leaching of the parent test substance but their testing is particularly required when the mixture is likely to affect the release rate (e.g. granular or controlled release formulations). Regarding mixture specific requirements for test design, it may be useful to consult with the regulatory authority prior to conducting the test. For aged residue leaching studies, the pure parent test substance should be used.

• 9.Before carrying out leaching tests in soil columns, the following information on the test chemical should preferably be available:

• (1) solubility in water [test method A.6] (13);

• (2) solubility in organic solvents;

• (3) vapour pressure [test method A.4] (13) and Henry's Law constant;

• (4) n-octanol/water partition coefficient [test methods A.8 and A.24] (13);

• (5) adsorption coefficient (Kd, Kf or KOC) [test methods C.18 and/or C.19] (13);

• (6) hydrolysis [test method C.7] (13);

• (7) dissociation constant (pKa) [OECD TG 112] (25);

• (8) aerobic and anaerobic transformation in soil [test method C.23] (13)

Note: The temperature at which these measurements were made should be reported in the respective test reports.

• 10.The amount of test chemical applied to the soil columns should be sufficient to allow for detection of at least 0,5 % of the applied dose in any single segment. For active chemicals in plant protection products, the amount of test chemical applied may correspond to the maximum recommended use rate (single application).

- 11.An appropriate analytical method of known accuracy, precision and sensitivity for the quantification of the test chemical and, if relevant, of its transformation products in soil and leachate must be available. The analytical detection limit for the test chemical and its significant transformation products (normally at least all transformation products ≥ 10 % of applied dose observed in transformation pathway studies, but preferably any relevant transformation products of concern) should also be known (see paragraph 17).

REFERENCE CHEMICALS

• 12.Reference chemicals with known leaching behaviour such as atrazine or monuron which can be considered moderate leachers in the field should be used for evaluating the relative mobility of the test chemical in soil (1)(8)(11). A nonsorbing and non degradable polar reference chemical (e.g. tritium, bromide, fluorescein, eosin) to trace the movement of water in the column may also be useful to confirm the hydrodynamic properties of the soil column.

- 13.Analytical standard chemicals may also be useful for the characterisation and/or identification of transformation products found in the soil segments and in the leachates by chromatographic, spectroscopic or other relevant methods.

DEFINITIONS AND UNITS

- 14.See Appendix 1.

QUALITY CRITERIA

Recovery

- 15.The sum of the percentages of the test chemical found in the soil segments and the column leachate after leaching gives the recovery for a leaching experiment. Recoveries should range from 90 % to 110 % for radio-labelled chemicals (11) and from 70 % to 110 % for non-labelled chemicals (8).

Repeatability and sensitivity of analytical method

• 16.Repeatability of the analytical method to quantify test chemical and transformation products can be checked by duplicate analysis of the same extract of a soil segment or of a leachate (see paragraph 11).

- 17.The limit of detection (LOD) of the analytical method for the test chemical and for the transformation products should be at least 0,01 mg · kg^{- 1} in each soil segment or leachate (as test chemical) or 0,5 % of applied dose in any single segment whichever is lower. The limit of quantification (LOQ) should also be specified.

DESCRIPTION OF THE TEST METHOD

Test system

• 18.Leaching columns (sectionable or non-sectionable) made of suitably inert material (e.g. glass, stainless steel, aluminium, teflon, PVC, etc.) with an inner diameter of at least 4 cm and a minimum height of 35 cm are used for the test. Column materials should be tested for potential interactions with the test chemical and/or its transformation products. Examples of suitable sectionable and non-sectionable columns are shown in Appendix 2.

• 19.Spoon, plunger and vibration apparatus are used for filling and packing the soil columns.

- 20.For application of artificial rain to the soil columns, piston or peristaltic pumps, showering heads, Mariotte bottles or simple dropping funnels can be used.

Laboratory equipment and chemicals

• 21.Standard laboratory equipment is required, in particular the following:

• (1) analytical instruments such as GLC, HPLC and TLC equipment, including the appropriate detection systems for analysing labelled or unlabelled chemicals or inverse isotope dilution method;

• (2) instruments for identification purposes (e.g. MS, GC-MS, HPLC-MS, NMR, etc.);

• (3) liquid scintillation counter for radio-labelled test chemical;

• (4) oxidiser for combustion of labelled material;

• (5) extraction apparatus (for example, centrifuge tubes for cold extraction and Soxhlet apparatus for continuous extraction under reflux);

• (6) instrumentation for concentrating solutions and extracts (e.g. rotating evaporator).

- 22.Chemicals used include: organic solvents, analytical grade, such as acetone, methanol, etc.; scintillation liquid; 0,01 M CaCl2 solution in distilled or deionised water (= artificial rain).

Test chemical

• 23.To apply the test chemical to the soil column it should be dissolved in water (deionised or distilled). If the test chemical is poorly soluble in water, it can be applied either as formulated product (if necessary after suspending or emulsifying in water) or in any organic solvent. In case an organic solvent is used, it should be kept to a minimum and should be evaporated from the surface of the soil column prior to start of leaching procedure. Solid formulations, such as granules, should be applied in the solid form without water; to allow a better distribution over the surface of the soil column, the formulated product may be mixed with a small amount of quartz sand (e.g. 1 g) before application.

- 24.The amount of test chemical applied to the soil columns should be sufficient to allow for detection of at least 0,5 % of the applied dose in any single segment. For active chemicals in plant protection products, this may be based on the maximum recommended use rate (single application rate) and, for both parent and aged leaching, should be related to the surface area of the soil column used ([^E0233]).

Reference chemical

- 25.A reference chemical should be used in the leaching experiments (see paragraph 12). It should be applied to the soil column surface in a similar way as the test chemical and at an appropriate rate that enables adequate detection either as an internal standard together with the test chemical on the same soil column or alone on a separate soil column. It is preferred that both chemicals be run on the same column, except when both chemicals are similarly labelled.

Soils

Soil selection

- 26.For leaching studies with the parent test chemical 3 to 4 soils with varying pH, organic carbon content and texture should be used (12). Guidance for selection of soils for leaching experiments is given in Table 1 below. For ionisable test chemicals the selected soils should cover a wide range of pH, in order to evaluate the mobility of the chemical in its ionised and unionised forms; at least 3 soils should have a pH at which the test chemical is in its mobile form.

Table 1

Guidance for selection of soils for leaching studies

Soil No.	pH value	Organic carbon %	Clay content %	Texture [^E0234]
1	> 7,5	3,5 - 5,0	20 - 40	clay loam
2	5,5 - 7,0	1,5 - 3,0	15 - 25	silt loam
3	4,0 - 5,5	3,0 - 4,0	15 - 30	loam
4	< 4,0 - 6,0 [^E0235]	< 0,5 - 1,5 [^E0235] [^E0236]	< 10 - 15 [^E0235]	loamy sand
5	< 4,5	> 10 [^E0237]	< 10	loamy sand/sand

According to FAO and USDA systems (14).

§  The respective variables should preferably show values within the range given. If, however, difficulties in finding appropriate soil material occur, values below the indicated minimum are accepted.

‡  Soils with less than 0,3 % organic carbon may disturb correlation between organic content and adsorption. Thus, it is recommended to use soils with a minimum organic carbon content of 0,3 %.

Soils with very high carbon content (e.g. > 10 %) may not be acceptable legally e.g. for pesticide registration purposes.

• 27.Other soil types may sometimes be necessary to represent cooler, temperate and tropical regions. Therefore, if other soil types are preferred, they should be characterised by the same parameters and should have similar variations in properties as those described in the guidance for selection of soils for leaching studies (see Table 1 above), even if they do not match the criteria exactly.

• 28.For leaching studies with ‘aged residues’, one soil should be used (12). It should have a sand content > 70 % and an organic carbon content between 0,5 - 1,5 % (e.g. soil No. 4 in Table 1). Use of more soil types may be necessary if data on the transformation products are important.

- 29.All soils should be characterised at least for texture [% sand, % silt, % clay according to FAO and USDA classification systems (14)], pH, cation exchange capacity, organic carbon content, bulk density (for disturbed soil) and water holding capacity. Measurement of microbial biomass is only required for the soil which is used in the ageing/incubation period carried out before the aged leaching experiment. Information on additional soil properties (e.g. soil classification, clay mineralogy, specific surface area) may be helpful for interpreting the results of this study. For determination of soil characteristics the methods recommended in references (15)(16)(17)(18)(19) can be used.

Collection and storage of soils

• 30.The soils should be taken from the top layer (A-horizon) to a maximum depth of 20 cm. Remains of vegetation, macro-fauna and stones should be removed. The soils (except those used for ageing the test chemical) are air-dried at room temperature (preferably between 20-25 C). Disaggregation should be performed with minimal force, so that the original texture of the soil will be changed as little as possible. The soils are sieved through a ≤ 2 mm sieve. Careful homogenisation is recommended, as this enhances the reproducibility of the results. Before use the soils can be stored at ambient temperature and kept air dried (12). No limit on storage time is recommended but soils stored for more than 3 years should be re-analysed prior to use with respect to their organic carbon content and pH.

- 31.Detailed information on the history of the field sites from where the test soils are collected should be available. Details include exact location [exactly defined by UTM (Universal Transversal Mercator-Projection/European Horizontal Datum) or geographical co-ordinates], vegetation cover, treatments with crop protection chemicals, treatments with organic and inorganic fertilisers, additions of biological materials or accidental contamination (12). If soil has been treated with the test chemical or its structural analogues within the previous four years, these soils should not be used for leaching studies.

Test conditions

• 32.During the test period, the soil leaching columns should be kept in the dark at ambient temperature as long as this temperature is maintained within a range of ± 2 °C. Recommended temperatures are between 18 and 25 °C.

- 33.Artificial rain (0,01 M CaCl2) should be applied continuously to the surface of the soil columns at a rate of 200 mm over a period of 48 hours ([^E0238]); this rate is equivalent to an application of 251 ml for a column with an inner diameter of 4 cm. If needed for the purpose of the test, other rates of artificial rainfall and longer duration may additionally be used.

Performance of the test

Leaching with parent test chemical

• 34.At least duplicate leaching columns are packed with untreated, air-dried and sieved soil (< 2 mm) up to a height of approximately 30 cm. To obtain uniform packing, the soil is added to the columns in small portions with a spoon and pressed with a plunger under simultaneous gentle column vibration until the top of the soil column does not sink in further. Uniform packing is required for obtaining reproducible results from leaching columns. For details on column packing techniques, see references (20) (21) and (22). To control the reproducibility of the packing procedure, the total weight of the soil packed in the columns is determined ([^E0239]); the weights of the duplicate columns should be similar.

• 35.After packing, the soil columns are pre-wetted with artificial rain (0,01 M CaCl2) from bottom to top in order to displace the air in the soil pores by water. Thereafter the soil columns are allowed to equilibrate and the excess water is drained off by gravity. Methods for column saturation are reviewed in reference (23).

• 36.Then the test chemical and/or the reference chemical are applied to the soil columns (see also paragraphs 23-25). To obtain a homogeneous distribution the solutions, suspensions or emulsions of the test and/or reference chemical should be applied evenly over the surface of the soil columns. If incorporation into soil is recommended for the application of a test chemical, it should be mixed in a small amount (e.g. 20 g) of soil and added to the surface of the soil column.

• 37.The surfaces of the soil columns are then covered by a glass sinter disk, glass pearls, glass fibre filters or a round filter paper to distribute the artificial rain evenly over the entire surface and to avoid disturbance of the soil surface by the rain drops. The larger the column diameter the more care is needed for the application of the artificial rain to the soil columns to ensure an even distribution of the artificial rain over the soil surface. Then the artificial rainfall is added to the soil columns drop-wise with the aid of a piston or a peristaltic pump or a dropping funnel. Preferably, the leachates should be collected in fractions and their respective volumes are recorded ([^E0240]).

- 38.After leaching and allowing the columns to drain, the soil columns are sectioned in an appropriate number of segments depending on the information required from the study, the segments are extracted with appropriate solvents or solvent mixtures and analysed for the test chemical and, when appropriate, for transformation products, for total radioactivity and for the reference chemical. The leachates or leachate fractions are analysed directly or after extraction for the same products. When radio-labelled test chemical is used, all fractions containing ≥ 10 % of the applied radioactivity should be identified.

Leaching with aged residues

• 39.Fresh soil (not previously air-dried) is treated at a rate corresponding to the surface area of the soil columns (see paragraph 24) with the radio-labelled test chemical and incubated under aerobic conditions according to Test Method C.23 (13). The incubation (ageing) period should be long enough to produce significant amounts of transformation products; an ageing period of one half-life of the test chemical is recommended ([^E0241]), but should not exceed 120 days. Prior to leaching, the aged soil is analysed for the test chemical and its transformation products.

• 40.The leaching columns are packed up to a height of 28 cm with the same soil (but air-dried) as used in the ageing experiment as described in paragraph 34 and the total weight of the packed soil columns is also determined. The soil columns are then pre-wetted as described in paragraph 35.

• 41.Then the test chemical and its transformation products are applied to the surface of the soil columns in the form of aged soil residues (see paragraph 39) as a 2 cm soil segment. The total height of the soil columns (untreated soil + aged soil) should preferably not exceed 30 cm (see paragraph 34).

• 42.The leaching is carried out as described in paragraph 37.

- 43.After leaching, soil segments and leachates are analysed as indicated in paragraph 38 for the test chemical, its transformation products and not-extracted radioactivity. To determine how much of the aged residue is retained in the top 2-cm layer after leaching, this segment should be analysed separately.

DATA AND REPORTING

Treatment of results

• 44.The amounts of test chemical, transformation products, non-extractables and, if included, of the reference chemical should be given in % of applied initial dose for each soil segment and leachate fraction. A graphical presentation should be given for each column plotting the percentages found as a function of the soil depths.

• 45.When a reference chemical is included in these column leaching studies, the leaching of a chemical can be evaluated on a relative scale using relative mobility factors (RMF; for definition see Appendix 3) (1)(11) which allows the comparison of leaching data of various chemicals obtained with different soil types. Examples of RMF-values for a variety of crop protection chemicals are given in Appendix 3.

- 46.Estimates of Koc (organic carbon normalised adsorption coefficient) and Kom (organic matter normalised distribution coefficient) can also be obtained from column leaching results by using average leaching distance or established correlations between RMF and Kom respectively Koc (4) or by applying simple chromatographic theory (24). However, the latter method should be used with caution especially when considering that the leaching process does not solely involve saturated flow conditions, but rather unsaturated systems.

Interpretation of results

- 47.The column leaching studies described in this method allow determining the leaching or mobility potential in soil of the test chemical (in the parent leaching study) and/or its transformation products (in the aged residue leaching study). These tests do not quantitatively predict leaching behaviour under field conditions, but they can be used to compare the ‘leachability’ of one chemical with others whose leaching behaviour may be known (24). Likewise, they do not quantitatively measure the percentage of applied chemical that might reach ground water (11). However, the results of column leaching studies may assist in deciding whether additional semi-field or field testing has to be carried out for chemicals showing a high mobility potential in laboratory tests.

Test report

• 48.The report must include:

Test chemical and reference chemical (when used):

• — common name, chemical name (IUPAC and CAS nomenclature), CAS number, chemical structure (indicating position of label when radio-labelled material is used) and relevant physical-chemical properties;

• — purities (impurities) of test chemical;

• — radiochemical purity of labelled chemical and specific activity (where appropriate).

Test soils:

• — details of collection site;

• — properties of soils, such as pH, organic carbon and clay content, texture and bulk density (for disturbed soil);

• — soil microbial activity (only for soil used for ageing of test chemical);

• — length of soil storage and storage conditions.

Test conditions:

• — dates of the performance of the studies;

• — length and diameter of leaching columns;

• — total soil weight of soil columns;

• — amount of test chemical and, if appropriate, reference chemical applied;

• — amount, frequency and duration of application of artificial rain;

• — temperature of experimental set-up;

• — number of replications (at least two);

• — methods for analysis of test chemical, transformation products and, where appropriate, of reference chemical in the various soil segments and leachates;

• — methods for the characterisation and identification of transformation products in the soil segments and leachates.

Test results:

• — tables of results expressed as concentrations and as % of applied dose for soil segments and leachates;

• — mass balance, if appropriate;

• — leachate volumes;

• — leaching distances and, where appropriate, relative mobility factors;

• — graphical plot of % found in the soil segments versus depth of soil segment;

- — discussion and interpretation of results.

LITERATURE

• (1)Guth, J.A., Burkhard, N. and Eberle, D.O. (1976). Experimental Models for Studying the Persistence of Pesticides in Soil. Proc. BCPC Symposium: Persistence of Insecticides and Herbicides.

• (2)Russel, M.H. (1995). Recommended approaches to assess pesticide mobility in soil. In progress in Pesticide Biochemistry and Toxicology, Vol. 9 (Environmental Behaviour of Agrochemicals — T.R. Roberts and P.C. Kearney, Eds.). J. Wiley & Sons.

• (3)Briggs, G.G. (1981). Theoretical and experimental relationships between soil adsorption, octanol-water partition coefficient, water solubilities, bioconcentration factors, and the parachor. J.Agric. Food Chem. 29, 1050-1059.

• (4)Chiou, C.T., Porter, P.E. and Schmedding, D.W. (1983). Partition equilibria of non-ionic organic compounds between soil organic matter and water. Environ. Sci. Technol. 17, 227-231.

• (5)Guth, J.A. (1983). Untersuchungen zum Verhalten von Pflanzenschutzmitteln im Boden. Bull. Bodenkundliche Gesellschaft Schweiz 7, 26-33.

• (6)US-Environmental Protection Agency (1982). Pesticide Assessment Guidelines, Subdivision N. Chemistry: Environmental Fate.

• (7)Agriculture Canada (1987). Environmental Chemistry and Fate Guidelines for registration of pesticides in Canada.

• (8)Annex I to Commission Directive 95/36/EC of 14 July 1995 amending Council Directive 91/414/EEC concerning the placing of plant protection products on the market, OJ L 172, 22.7.1995, p. 8.

• (9)Dutch Commission for Registration of Pesticides (1991). Application for registration of a pesticide. Section G: Behaviour of the product and its metabolites in soil, water and air.

• (10)BBA (1986). Richtlinie für die amtliche Prüfung von Pflanzenschutzmitteln, Teil IV, 4-2. Versickerungsverhalten von Pflanzenschutzmitteln.

• (11)SETAC (1995). Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides. Mark R. Lynch, Ed.

• (12)OECD (1995). Final Report of the OECD Workshop on Selection of Soils/Sediments. Belgirate, Italy, 18-20 January 1995.

• (13)The following chapters of this Annex:

Chapter A.4, vapour pressure

Chapter A.6, Water solubility

Chapter A.8, Partition coefficient, shake flask method

Chapter A.24, Partition coefficient, HPLC method

Chapter C.7, degradation — abiotic degradation: hydrolysis as a function of pH

Chapter C.18, Adsorption/desorption using a batch equilibrium method

Chapter C.23, Aerobic and anaerobic transformation in soil

• (14)Soil Texture Classification (US and FAO systems). Weed Science, 33, Suppl. 1 (1985) and Soil Sci. Soc. Amer. Proc. 26, 305 (1962).

• (15)Methods of Soil Analysis (1986). Part 1, Physical and Mineralogical Methods (A. Klute, Ed.). Agronomy Series No. 9, 2nd Edition.

• (16)Methods of Soil Analysis (1982). Part 2, Chemical and Microbiological Properties (A.L. Page, R.H. Miller and D.R. Kelney, Eds.). Agronomy Series No. 9, 2nd Edition.

• (17)ISO Standard Compendium Environment (1994). Soil Quality — General aspects; chemical and physical methods of analysis; biological methods of analysis. First Edition.

• (18)Mückenhausen, E. (1975). Die Bodenkunde und ihre geologischen, geomorphologischen, mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt/Main.

• (19)Scheffer, F. and Schachtschabel, P. (1998). Lehrbuch der Bodenkunde. F. Enke Verlag, Stuttgart.

• (20)Weber, J.B. and Peeper, T.F. (1977). In Research Methods in Weed Science, 2nd Edition (B. Truelove, Ed.). Soc. Weed Sci., Auburn, Alabama, 73-78.

• (21)Weber, J.B., Swain, L.R., Strek, H.J. and Sartori, J.L. (1986). In Research Methods in Weed Science, 3rd Edition (N.D. Camper, Ed.). Soc. Weed Sci., Champaign, IL, 190-200.

• (22)Oliveira, et al. (1996). Packing of sands for the production of homogeneous porous media. Soil Sci. Soc. Amer. J. 60(1): 49-53.

• (23)Shackelford, C. D. (1991). Laboratory diffusion testing for waste disposal. — A review. J. Contam. Hydrol. 7, 177-217.

• (24)(Hamaker, J.W. (1975). Interpretation of soil leaching experiments. In Environmental Dynamics of Pesticides (R. Haque, V.H. Freed, Eds), 115-133. Plenum Press, New York.

- (25)OECD (1981). Dissociation constants in water. OECD Guideline for Testing of Chemicals, No. 4112, OECD, Paris

Appendix 1

Definitions and units

Aged soil residue : Test chemical and transformation products present in soil after application and following a period long enough to allow transport, adsorption, metabolism, and dissipation processes to alter the distribution and chemical nature of some of the applied chemical (1).

Artificial rain : 0,01 M CaCl2 solution in distilled or deionised water.

Average Leaching Distance : Bottom of soil section where cumulative recovered chemical = 50 % of total recovered test chemical [normal leaching experiment], or; (bottom of soil section where cumulative recovered chemical = 50 % of total recovered test chemical) — ((thickness of aged residue layer)/2) [aged residue leaching study]

Chemical : a substance or a mixture.

Leachate : Aqueous phase percolated through a soil profile or a soil column (1).

Leaching : Process by which a chemical moves downward through the soil profile or a soil column (1).

Leaching distance : Deepest soil segment in which ≥ 0,5 % of the applied test chemical or aged residue was found after the leaching process (equivalent to penetration depth).

Limit of detection (LOD) and limit of quantification (LOQ) : The limit of detection (LOD) is the concentration of a chemical below which the identity of the chemical cannot be distinguished from analytical artefacts. The limit of quantification (LOQ) is the concentration of a chemical below which the concentration cannot be determined with an acceptable accuracy.

RMF Relative Mobility Factor : (leaching distance of test chemical (cm))/(leaching distance of reference chemical (cm))

Test chemical : Any substance or mixture tested using this test method.

Transformation product : All chemicals resulting from biotic or abiotic transformation reactions of the test chemical including CO2 and products that are bound in residues.

Soil

:

A mixture of mineral and organic chemical constituents, the latter containing compounds of high carbon and nitrogen content and of high molecular weights, populated by small (mostly micro-) organisms. Soil may be handled in two states:

• — undisturbed, as it has developed with time, in characteristic layers of a variety of soil types;

• — disturbed, as it is usually found in arable fields or as occurs when samples are taken by digging and used in this test method (2).

• (1)Holland, P.T. (1996). Glossary of Terms Relating to Pesticides. IUPAC Reports on Pesticide (36). Pure & Appl. Chem. 68, 1167-1193.

- (2)OECD Test Guideline 304 A: Inherent Biodegradability in Soil (adopted 12 May 1981).

Appendix 2

Figure 1

Example of non- sectionable leaching columns made of glass

With a length of 35 cm and an inner diameter of 5 cm (1)

Image omitted; see official source

Text of image

← Glass wool plug to keep the soil in the column

← Layer of quartz sandsurface

← Glass column filled with test soil (when testing photolabile products columns should be wrapped in aluminium foil)

← Glass sinter disc to avoid disturbance of the soil surface and to evenly distribute the artificial rain

← Dropping funnels for application of artificial rain

← Round-bottom flask for collection of leachate; wrapped in aluminium foil to exclude photolysis

- (1)Drescher, N. (1985). Moderner Acker- und Pflanzenbau aus Sicht der Pflanzenschutzmittelindustrie. In Unser Boden — 70 Jahre Agrarforschung der BASF AG, 225-236. Verlag Wissenschaft und Politik, Köln.

Figure 2

Example of a sectionable metal column with 4 cm inner diameter (1)

Image omitted; see official source

- (1)Burkhard, N., Eberle D.O. and Guth, J.A. (1975). Model systems for studying the environmental behaviour of pesticides. Environmental Quality and Safety, Suppl. Vol. III, 203-213.

Appendix 3

Examples of Relative Mobility Factors [^E0242] (RMF) for a variety of Crop protection chemicals (1)(2) and corresponding mobility classes [^E0244]

RMF-Range	Chemical (RMF)	Mobility Class
≤ 0,15	Parathion (< 0,15), Flurodifen (0,15)	I immobile
0,15 - 0,8	Profenophos (0,18), Propiconazole (0,23), Diazinon (0,28), Diuron (0,38), Terbuthylazine (0,52), Methidathion (0,56), Prometryn (0,59), Propazine (0,64), Alachlor (0,66), Metolachlor (0,68)	II slightly mobile
0,8 - 1,3	Monuron [^E0243] (1,00), Atrazine (1,03), Simazine (1,04), Fluometuron (1,18)	III moderately mobile
1,3 - 2,5	Prometon (1,67), Cyanazine (1,85), Bromacil (1,91), Karbutilate (1,98)	IV fairly mobile
2,5 - 5,0	Carbofuran (3,00), Dioxacarb (4,33)	V mobile
> 5,0	Monocrotophos (> 5,0), Dicrotophos (> 5,0)	VI very mobile

The Relative Mobility Factor is derived as follows (3): Image omitted; see official source

Reference chemical

+  Other systems to classify a chemical's mobility in soil are based on Rf values from soil thin-layer chromatography (4) and on Koc values (5)(6).

• (1)Guth, J.A. (1985). Adsorption/desorption. In Joint International Symposium ‘Physicochemical Properties and their Role in Environmental Hazard Assessment’. Canterbury, UK, 1-3 July 1985.

• (2)Guth, J.A. and Hörmann, W.D. (1987). Problematik und Relevanz von Pflanzenschutzmittel-Spuren im Grund (Trink-) Wasser. Schr.Reihe Verein WaBoLu, 68, 91-106.

• (3)Harris, C.I. (1967). Movement of herbicides in soil. Weeds 15, 214-216.

• (4)Helling, C.S. (1971). Pesticide mobility in soils. Soil Sci. Soc. Am. Proc. 35, 743-748.

• (5)McCall, P.J., Laskowski, D.A., Swann, R.L. and Dishburger, H.J. (1981). Measurements of sorption coefficients of organic chemicals and their use in environmental fate analysis. In Test Protocols for Environmental Fate and Movement of Toxicants. Proceedings of AOAC Symposium, AOAC, Washington D.C.

- (6)Hollis, J.M. (1991). Mapping the vulnerability of aquifers and surface waters to pesticide contamination at the national/regional scale. BCPC Monograph No. 47 Pesticides in Soil and Water, 165-174.

C.45.   ESTIMATION OF EMISSIONS FROM PRESERVATIVE — TREATED WOOD TO THE ENVIRONMENT: LABORATORY METHOD FOR WOODEN COMMODITIES THAT ARE NOT COVERED AND ARE IN CONTACT WITH FRESH WATER OR SEAWATER

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 313 (2007). The emissions from preservative-treated wood to the environment need to be quantified to enable an environmental risk assessment of the treated wood. This test method describes a laboratory method for the estimation of emissions from preservative-treated wood in two situations where emissions could enter the environment:

• — Emissions from treated wood in contact with fresh water. Emissions from the surface of the treated wood could enter the water.

• — Emissions from treated wood in contact with seawater. Emissions from the surface of the treated wood could enter the seawater.

• 2.This test method is intended for testing the emissions from wood and wooden commodities that are not covered and are in contact with fresh water or seawater. Use Classes are used internationally and categorise the biological hazard to which the treated commodity will be subjected. Use Classes also define the situation in which the treated commodity is used and determine the environmental compartments (air, water, soil) which are potentially at risk from the preservative treated wood.

• 3.The test method is a laboratory procedure for obtaining samples (emissate) from water used to immerse treated wood, at increasing time intervals after exposure. The quantity of emissions in the emissate is related to the surface area of the wood and the length of exposure, to estimate a flux in mg/m²/day. The flux (leaching rate) after increasing periods of exposure can thus be estimated.

- 4.The quantity of emissions can be used in an environmental risk assessment of the treated wood.

INITIAL CONSIDERATIONS

• 5.The mechanism of leaching at the wood surface by fresh water is not assumed to be identical in nature and severity to leaching from a wood surface by seawater. Thus, for wood preservative products or mixtures used to treat wood used in seawater environs, a wood leaching study for seawater is necessary.

• 6.The wood, in the case of wood treated with a wood preservative, should be representative of commercially used wood. It should be treated in accordance with the preservative manufacturer's instructions and in compliance with appropriate standards and specifications. The parameters for the post treatment conditioning of the wood prior to the commencement of the test should be stated.

• 7.The wood samples used should be representative of the commodities used (e.g., with regard to species, density and other characteristics).

• 8.The test can be applied to wood using a penetrating process or superficial application or to treated wood which has an additional mandatory surface treatment (e.g., paint that is applied as a requirement for commercial use).

- 9.The composition, amount, pH and the physical form of water is important in determining the quantity, content and nature of emissions from wood.

PRINCIPLE OF THE TEST METHOD

• 10.Preservative-treated wood test specimens are immersed in water. The water (emissate) is collected and chemically analysed multiple times over the exposure period sufficient to perform statistical calculations. Emission rates in mg/m²/day are calculated from analytical results. The sampling periods should be recorded. Tests with untreated samples can be discontinued if there is no background detected in the first three data points.

- 11.The inclusion of untreated wood specimens allows for the determination of background levels for emissates from wood other than the preservative used.

QUALITY CRITERIA

Accuracy

• 12.The accuracy of the test method to estimate emission depends upon the test specimens being representative of commercially treated wood, how representative the water is of real water and how the exposure regime is representative of natural conditions.

- 13.The accuracy, precision and repeatability of the analytical method should be determined before conducting the test.

Reproducibility

- 14.Three water samples are collected and analysed and the mean value is taken as the emission value. The reproducibility of the results within one laboratory and between different laboratories depends upon the immersion regime and the wood used as test specimens.

Acceptable Range of Results

- 15.A range of results from this test where the upper and lower values differ by less than one order of magnitude is acceptable.

TEST CONDITIONS

Water

• 16.Freshwater leaching scenarios: Deionised water (e.g., ASTM D 1193 Type II) is recommended for use in the leaching test when wood exposed to freshwater is to be evaluated. The water temperature shall be 20 °C +/– 2 °C and the measured pH and water temperature included in the test report. Analysis of samples of the water used taken before immersion of the treated specimens allows the estimation of the analysed chemicals in the water. This is a control to determine background levels of chemicals which are then chemically analysed.

- 17.Seawater leaching scenarios: Synthetic seawater (e.g., ASTM D 1141 Substitute Ocean Water, without Heavy Metals) is recommended for use in the leaching test when wood exposed to seawater is to be evaluated. The water temperature shall be 20 °C +/– 2 °C and the measured pH and water temperature included in the test report. Analysis of samples of the water used taken before immersion of the treated specimens allows the estimation of the analysed chemicals in the water. This is a control for the analysis of background levels for chemicals of importance.

Wood Test Specimens

• 18.The wood species should be typical of the wood species used for the efficacy testing of wood preservatives. The recommended species are Pinus sylvestris L. (Scots pine), Pinus resinosa Ait. (red pine) or Pinus spp (Southern pine). Additional tests may be made using other species.

• 19.Straight grained wood without knots should be used. Material of a resinous appearance should be avoided. The wood should be typical of wood which is available commercially. The source, density and number of annual rings per 10 mm should be recorded.

• 20.Wood test specimens are recommended to be sets of five according to EN 113 size blocks (25 mm × 50 mm × 15 mm dimensions) with the longitudinal faces parallel to the grain of the wood, although other dimensions such as 50 mm, by 150 mm, by 10 mm may be used. The test specimen should be completely immersed into the water. Test specimens shall consist of 100 % sapwood. Each specimen is uniquely marked so that it can be identified throughout the test.

• 21.All test specimens should be planed or plane sawn and the surfaces should not be sanded.

• 22.The number of sets of wood test specimens used for analysing is at least five: three sets of specimens are treated with preservative, one set of specimens is untreated and one set of specimens for the estimation of the oven dry moisture content of the test specimens before treatment. Sufficient test specimens are prepared to allow selection of three sets of specimens which are within 5 % of the mean value of the preservative retentions of the pool of test specimens.

• 23.All test specimens are end-sealed with a chemical which prevents penetration of preservative into the end grain of the specimens or prevents leaching from the specimens via the end grain. It is necessary to distinguish between specimens used for superficial application and penetration processes for the application of the end-sealant. The application of the end-sealant has to be applied prior to treatment only in case of superficial application.

- 24.The end-grain has to be open for treatments by penetration processes. Therefore, the specimens have to be end-sealed at the end of the conditioning period. The emission has to be estimated for the longitudinal surface area only. Sealants should be inspected and reapplied if necessary prior to initiating leaching and should not be reapplied after leaching has been initiated.

Immersion Container

- 25.The container is made of an inert material and is large enough to contain 5 EN113 wood specimens in 500 ml of water resulting in a surface area to water volume ratio of 0,4 cm²/ml.

Specimen Test Assembly

- 26.The test specimens are supported on an assembly which allows all exposed surfaces of the specimen to be in contact with water.

PROCEDURE FOR PRESERVATIVE TREATMENT

Preparation of the Treated Test Specimens

- 27.The wood test specimen to be treated with the preservative under test is treated by the method specified for the preservative, which may be by a penetrating treatment process or a superficial application process, which may be with a dip, spray or brush.

Preservatives to be applied by penetrating treatment process

• 28.A solution of the preservative should be prepared that will achieve the specified uptake or retention when applied using the penetrating treatment process. The wood test specimen is weighed and its dimensions are measured. The penetrating treatment process should be as specified for the application of the preservative to wood for use in Use Class 4 or 5. The specimen is again weighed after treatment and the retention of the preservative (kg/m³) is calculated from the equation:

Image omitted; see official source

- 29.Note that timber treated in an industrial treatment plant (e.g. by vacuum pressure impregnation) may be used in this test. The procedures used should be recorded and the retention of material treated in this way must be analysed and recorded.

Preservatives to be applied by superficial application processes

• 30.The superficial application process includes dipping, spraying or brushing of the wood test specimens. The process and application rate (e.g. litres/m²) should be as specified for the superficial application of the preservative.

- 31.Also note in this case, timber treated in an industrial treatment plant may be used in this test. The procedures used should be recorded and the retention of material treated in this way must be analysed and recorded.

Conditioning of the Test Specimens after Treatment

- 32.After treatment, the treated test specimens should be conditioned in accordance with the recommendations made by the supplier of the test preservative according to the preservative label requirements or as in accordance with commercial treatment practices or in accordance with EN 252 Standard.

Preparation and Selection of Test Specimens

- 33.After post treatment conditioning, the mean retention of the group of test specimens is calculated and three representative sets of specimens with a retention within 5 % of the mean for the group are randomly selected for leaching measurements.

PROCEDURE FOR PRESERVATIVE EMISSION MEASUREMENTS

Immersion Method

• 34.The test specimens are weighed and subsequently totally immersed in the water and the date and time recorded. The container is covered to reduce evaporation.

• 35.The water is replaced at the following intervals: 6 hours, 1 day, 2 days, 4 days, 8 days, 15 days, 22 days, 29 days (note: these are total times not interval times). The time and date of the water change and the mass of water recovered from the container should be recorded.

• 36.After each water exchange, a sample of water in which the set of test specimens has been immersed is retained for subsequent chemical analysis.

- 37.The sampling procedure allows the calculation of the profile of the quantity of emissions against time. Samples should be stored under conditions that preserve the analyte e.g., in a refrigerator in the dark to reduce microbial growth in the sample before analysis.

EMISSION MEASUREMENTS

Treated Samples

- 38.Collected water is chemically analysed for the active substance and/or relevant degradation/transformation products, if appropriate.

Untreated Samples

- 39.Collection of the water (emissate) in this system and subsequent analysis of chemicals that had leached from the untreated wood samples allow the estimation of the possible emission rate of the preservative from untreated wood. Collection and analysis of the emissate after increasing time periods of exposure allow the rate of change of the emission rate with time to be estimated. This analysis is a control procedure to determine background levels of the test chemical in untreated wood to confirm that the wood used as a source of samples had not been previously treated with the preservative.

DATA AND REPORTING

Chemical Analyses

- 40.The collected water is chemically analysed and the water analysis result is expressed in appropriate units, e.g., μg/l.

Reporting of Data

• 41.All results are recorded. The Appendix shows an example of a suggested recording form for one set of treated test specimens, and the summary table for calculating the mean emission values over each sampling interval.

- 42.The daily emission flux in mg/m²/day is calculated by taking the mean of the three measurements from the three replicates and dividing by the number of days of immersion.

Test Report

• 43.At least the following information shall be provided in the test report:

• — The name of the supplier of the preservative under test;

• — The specific and unique name or code of the preservative tested;

• — The trade or common name of the active ingredient(s) with a generic description of the co-formulants (e.g. co-solvent, resin), and the composition in % m/m of the ingredients;

• — The relevant retention or loading (in kg/m³ or l/m², respectively) specified for wood used in contact with water;

• — The species of wood used, with its density, and growth rate in rings per 10 mm;

• — The loading or retention of the preservative tested and the formula used to calculate the retention, expressed as l/m² or kg/m³;

• — The method of application of the preservative, specifying the treatment schedule used for a penetrating process, and the method of application if a superficial treatment was used;

• — The date of application of the preservative, and an estimate of the moisture content of the test specimens, expressed as a percentage;

• — Conditioning procedures used, specifying the type, conditions and duration;

• — Specification of the end sealant used and the number of times applied;

• — Specification of any subsequent treatment of the wood, e.g. specification of the supplier, type, characteristics and loading of a paint;

• — The time and date of each immersion event, the amount of water used for the immersion of the test specimens at each event, and the amount of water absorbed by the wood during immersion;

- — Any variation from the described method and any factors that may have influenced the results.

LITERATURE

• (1)European Standard, EN 84 — 1997. Wood preservatives. Accelerated ageing of treated wood prior to biological testing. Leaching procedure.

• (2)European Standard, EN 113/A1 — 2004. Wood preservatives. Test method for determining the protective effectiveness against wood destroying basidiomycetes. Determination of the toxic values.

• (3)European Standard, EN 252 — 1989. Field test method for testing the relative protective effectiveness of a wood preservative in ground contact.

• (4)European Standard, EN 335 — Part 1: 2006. Durability of wood and wood-based products — Definition of use classes — Part1: General.

• (5)American Society for Testing and Materials Standards, ASTM D 1141 — 1998. Standard Practice for the Preparation of Substitute Ocean Water, Without Heavy Metals. Annual Book of ASTM Standards, Volume 11.02.

- (6)American Society for Testing and Materials Standards, ASTM D 1193-77 Type II — 1983. Specifications for Reagent Water. Annual Book of ASTM Standards, Volume 11.01.

Appendix 1

Recording form for test method

Estimation of Emissions from Preservative-Treated Wood to the Environment: Laboratory Method for Wooden Commodities that are not Covered and are in Contact with Fresh Water or Seawater

Test house
Wood preservative	Wood preservative
Supplier of the preservative
Specific and unique name or code of the preservative
Trade or common name of the preservative
Co-formulants
Relevant retention for wood used in contact with water
Application	Application
Application method
Date of application
Formula used to calculate the retention:
Conditioning procedure
Duration of conditioning
End sealant/number of times applied
Subsequent treatment	if relevant
Test specimens	Test specimens
Wood species
Density of the wood	(minimum … mean value … maximum)
Growth rate (rings per 10 mm)	(minimum … mean value … maximum)
Moisture content
*Test assemblies* [^E0245]	Retention (e.g. kg/m3)
Treated ‘x’	Mean value and standard deviation or range for 5 specimens
Treated ‘y’	Mean value and standard deviation or range for 5 specimens
Treated ‘z’	Mean value and standard deviation or range for 5 specimens
Untreated
Variation of test method parameters	e.g. water quality, dimension of test specimens etc.

x, y, z represent the three replicate samples

Time	Water exchange	Specimen mass	Specimen mass	Water uptake	Water uptake	Water uptake	Water uptake	Water sample	Water sample	Water sample	Water sample	Water sample	Water sample	Water sample	Water sample	Water sample	Water sample	Water sample
Time	Water exchange	Treated (mean)	Untreated	Treated (mean)	Treated (mean)	Untreated	Untreated		Test water	Test water	Test water	Test water	x	x	y	y	z	z
	Date	g	g	g	g	g	g	no.	pH	pH	pH	pH	pH	pH	pH	pH	pH	pH
start
6h								1
24h								2
2 d								3
4 d								4
8 d								5
15 d								6
22 d								7
29 d								8

Please prepare separate tables for each active ingredient

Time	Water exchange	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results	Analytical Results
Time	Water exchange	Untreated specimens	Untreated specimens	Untreated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens	Treated specimens
Time	Water exchange	Concentration a.i. in water mg/l	Quantity emitted mg/m2	Emission rate mg/m2/d	Concentration a.i. in water	Concentration a.i. in water	Concentration a.i. in water	Concentration a.i. in water	Quantity emitted	Quantity emitted	Quantity emitted	Quantity emitted	Emission rate	Emission rate	Emission rate	Emission rate
Time	Water exchange	Concentration a.i. in water mg/l	Quantity emitted mg/m2	Emission rate mg/m2/d	x	y	z	Mean	x	y	z	Mean	x	y	z	Mean
	Date	Concentration a.i. in water mg/l	Quantity emitted mg/m2	Emission rate mg/m2/d	mg/l	mg/l	mg/l	mg/l	mg/m2	mg/m2	mg/m2	mg/m2	mg/m2/d	mg/m2/d	mg/m2/d	mg/m2/d
6h
24h
2 d
4 d
8 d
15 d
22 d
29 d

Note: Since results from untreated may have to be used to correct emission rates from treated samples, the untreated results should come first and all values for treated samples would be ‘corrected values’. There may also be a correction for the initial water analysis.

Appendix 2

Definitions

Chemical : A substance or a mixture.

Test chemical : Any substance or mixture tested using this test method.

C.46.   BIOACCUMULATION IN SEDIMENT-DWELLING BENTHIC OLIGOCHAETES

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 315 (2008) Sediment-ingesting endobenthic animals may be exposed to sediment bound substances (1). Among these sediment-ingesters, aquatic oligochaetes play an important role in the bottoms of the aquatic systems. They live in the sediment and often represent the most abundant species especially in habitats with environmental conditions adverse to other animals. By bioturbation of the sediment and by serving as prey these animals can have a strong influence on the bioavailability of such substances to other organisms, e.g. benthivorous fish. In contrast to epibenthic organisms, endobenthic aquatic oligochaetes burrow in the sediment, and ingest sediment particles below the sediment surface. Because of that, these organisms are exposed to substances via many uptake routes including direct contact, ingestion of contaminated sediment particles, porewater and overlying water. Some species of benthic oligochaetes that are currently used in ecotoxicological testing are described in Appendix 6.

• 2.The parameters which characterise the bioaccumulation of a substance include first of all the bioaccumulation factor (BAF), the sediment uptake rate constant (ks) and the elimination rate constant (ke). Detailed definitions of these parameters are provided in Appendix 1.

• 3.To assess the bioaccumulation potential of substances in general, and to investigate the bioaccumulation of substances which tend to partition into or onto the sediments, a compartment-specific test method is needed (1)(2)(3)(4).

• 4.This test method is designed to assess bioaccumulation of sediment-associated substances in endobenthic oligochaete worms. The test substance is spiked into the sediment. Using spiked sediment is intended to simulate a contaminated sediment.

• 5.This method is based on existing sediment toxicity and bioaccumulation test methods (1)(4)(5)(6)(7)(8)(9). Other useful documents are: the discussions and results of an international workshop (11), and the outcome of an international ring test (12).

- 6.This test applies to stable, neutral organic substances, which tend to associate with sediments. Bioaccumulation of sediment-associated, stable metallo-organic compounds can also be measured with this method (12). It is not applicable to metals and other trace elements (11) without modification of the test design with respect to substrate and water volumes, and possibly tissue sample size.

PREREQUISITE AND INFORMATION ON TEST SUBSTANCE